Ellen V. Rothenberg

Virtual memory T cells are increasingly recognized as a functionally distinct lineage within the CD8 T cell pool, but when and how commitment to the lineage is enforced remain poorly understood. Here we demonstrate that TVM lineage choice is exceptionally sensitive to dosage and repression competence of the key T cell transcription factor Bcl11b. Three different genetic models of slightly reduced Bcl11b each biased CD8 cell development to TVM generation without deregulating effector differentiation. Timed conditional knockouts and adoptive transfers narrowed the developmental window and showed that Bcl11b levels determine diversion to virtual memory fate uniquely during intrathymic positive selection. Whereas total Bcl11b loss disrupts TCR signalling, a <2-fold dose reduction of Bcl11b enhanced selective responses to TCR stimulation. Chromatin accessibility profiling and single cell RNA-seq indicated that Bcl11b dose reduction redirects cells to the TVM fate, from the late cycling fraction of mature CD8SP thymocytes, by a mechanism independent of previously described cytokine-driven pathways.

Virtual memory T cells are increasingly recognized as a functionally distinct lineage within the CD8 T cell pool, but when and how commitment to the lineage is enforced remain poorly understood. Here we demonstrate that T _VM lineage choice is exceptionally sensitive to dosage and repression competence of the key T cell transcription factor Bcl11b. Three different genetic models of slightly reduced Bcl11b each biased CD8 cell development to T _VM generation without deregulating effector differentiation. Timed conditional knockouts and adoptive transfers narrowed the developmental window and showed that Bcl11b levels determine diversion to virtual memory fate uniquely during intrathymic positive selection. Whereas total Bcl11b loss disrupts TCR signalling, a VM fate, from the late cycling fraction of mature CD8SP thymocytes, by a mechanism independent of previously described cytokine-driven pathways.

To access the earliest stages of murine T cell development, we adapted a serum-free expansion system for hematopoietic stem and progenitor-like cells. These cells undergo normal T cell differentiation in vivo and in vitro, verified by gene expression trajectories from single-cell RNA sequencing. Their absolute differentiation speed is slower than that of fresh progenitors but is modulated with Flt3L priming. Leveraging this system, the earliest T cell lineage–initiating events in response to Notch signaling included chromatin opening and transcriptional activation of the TCR-Cβ locus. Acute CRISPR knockouts revealed that some transcription factors normally inherited from bone marrow progenitors impede early T cell development, with differing effects on proliferation. Among 23 factors tested, Lmo2 knockout greatly accelerated the onset of germline TCR-Cβ locus transcription and expression of Tcf7 , Gata3 , and Runx1 and their targets. Thus, normal endogenous expression of this progenitor- and leukemia-associated factor markedly restrains T cell program initiation.

To gain access to the earliest stages of T cell development, we adapted a serum-free culture system that expands hematopoietic stem and progenitor-like cells. These expanded cells efficiently undergo normal T-cell differentiation in vivo and in vitro , verified by early gene expression trajectories from single-cell RNA sequencing, though their absolute differentiation speed is slower than that of fresh progenitors and can be modulated with cytokine priming. Leveraging this expansion system to observe the first T-lineage events, we revealed that initial Notch activation immediately induces chromatin opening and transcriptional activation of the TCR-Cβ locus. Additionally, acute CRISPR knockouts confirmed T-lineage entry requirements for Ikzf1 , Hes1 , Gabpa , and Myb while revealing that Lmo2 , Erg, Spi1, Hoxa9 , and Meis1 retard developmental progression with differing effects on proliferation. Endogenous expression of the stem, progenitor, and leukemia-associated factor Lmo2 markedly restrains initiation of the T cell program, with Lmo2 knockout greatly accelerating germline TCRβ locus transcription and expression of Tcf7, Gata3, Runx family, and E protein genes and their targets.

Pasadena, CA 91125 USA and 2 Jacques Loeb Centre for the History and Philosophy of Science, Ben-Gurion University of the Negev, Beer Sheva, Israel.The term "epigenetic" has had its meaning change substantially since it was proposed over 80 years ago (see, e.g., Morange 2002;Felsenfeld 2014;Deichmann 2016;Greally 2018).Originally conceived broadly as the set of processes that generate phenotype from genotype over time in development (Waddington 1942), it is currently used to mean the mechanisms demarcating different parts of the genome for differential gene activity in a given cellular context. We now have a highly sophisticated mechanistic understanding of the ways different genomic regions are marked by histone modification, DNA methylation, and chromatin remodelling in concert with differential local gene expression or local gene silencing. Other aspects of epigenetics include formation of dynamic protein condensates, chromatin looping, and shifts in intranuclear gene localization.All these epigenetic mechanisms themselves depend, directly or indirectly, on the activities of genes that encode chromatin modifying enzymes, and they gain their target-gene specificity by the trans-acting, sequence-specific transcription factors and regulatory RNAs, which guide the chromatin modifiers to specific genomic locations. Of particular interest is the reciprocal way that transcription factors and chromatin epigenetic states affect each other's action; sequence-specific transcription factor action is crucial to change gene expression for developmental progression, while chromatin states channel transcription factor activity and help to make development irreversible. This collection reveals some historical foundations of current chromatin research, and highlights the connection between events that are now called epigenetic, the genomic loci that encode epigenetic regulators, and the regulatory factors that guide them in development in different systems. It includes sophisticated case studies drawn from hematopoiesis, and an evaluation of the claims for epigenetic mechanisms in transgenerational inheritance in mammals.Deichmann (https://www.frontiersin.org/articles/10.3389/freae.2023.1334556/full) summarizes in-depth historical interviews with two pioneers of the field, Gary Felsenfeld and Sir Adrian Bird. Felsenfeld, who passed away at the beginning of May 2024, was a leader in many aspects of chromatin structural analysis. He specifically demonstrated the power of differential chromatin accessibility to identify active regulatory elements in the genome and investigated control of chromatin looping, the critical process that enables distal enhancers to interact with the correct promoters. Bird has long been a leader in the field of DNA methylation on CpG dinucleotides and their regulatory impact. This was one of the earliest-appreciated epigenetic marks, which first illustrated many of the paradigmatic aspects of epigenetic marking mechanisms in general. His work has revealed that a neurological disease, Rett syndrome, is caused by a mutation altering levels of a methyl-CpG binding protein that is an epigenetic modifier. As he noted, though: this syndrome is caused by mutations that change the DNA sequence. The primary change is the DNA sequence, and the secondary consequences are the epigenetic changes. It is thus an excellent example of the fact that genetics and epigenetics are closely linked. Some of the most sophisticated work on epigenetic causality has been carried out in hematopoietic systems. Even before the sequencing of mouse and human genomes, intense work was already under way to determine the epigenetic mechanisms that control highlyspecific expression of the genes that encode immunoglobulins in B cells and T-cell receptors in T cells (Nelsen et al. 1993;Stanhope-Baker et al. 1996;Nikolajczyk et al. 1999;Bergman et al. 2003;Cobb et al. 2006). This head start established these immunoreceptor loci as, arguably, the parts of the mammalian genome where epigenetic control is the best understood, as reviewed by Ma et al. (https://doi.org/10.3389/freae.2024.1489362 ).These loci undergo highly cell type-specific DNA-level rearrangement in development as well as transcriptional activation. However, the chromatin structures, enhancers, transcription factors that work on the enhancers, and impacts of enhancer mutations on transcription in the correct lineages at the correct developmental times have been defined functionally in all of these loci with exceptional clarity, setting precedents that have subsequently proven to be of much wider generality. These genes continue to provide important insights: they include strong evidence for transcription factor combinatorial action and multiple well-demonstrated cases of temporal mismatches between chromatin opening and enhancer activation leading to transcription. These imply that additional levels of regulation still need to be explained.Hematopoiesis more broadly has also been subjected to intense study to relate epigenetic mechanisms to developmentally controlled, lineage-specific cell type identity (e.g., Zeller et al. 2023;Yoshida et al. 2019;Goode et al. 2016;Javierre et al. 2016;Gonzalez et al. 2015).The research paper by Maytum et al.(https://www.frontiersin.org/articles/10.3389/freae.2024.1465958/full) takes a genomewide view of the epigenomic transformations that accompany the earliest emergence of hematopoiesis. The DNA sequences in regions of chromatin undergoing epigenetic change give evidence that sequence-specific factors of the AP-1 transcription factor family, which normally become active in cells responding to acute signaling, are involved in opening chromatin at specific sites marking different developmental pathways. A paradox is that AP-1 is not cell type-specific; how can it guide distinct patterns of epigenomic change for different cell types? The authors present evidence that AP-1 gains distinct target site specificities...

The rate at which cells enter the T cell pathway depends not only on the immigration of hematopoietic precursors into the strong Notch signaling environment of the thymus but also on the kinetics with which each individual precursor cell reaches T-lineage commitment once it arrives. Notch triggers a complex, multistep gene regulatory network in the cells in which the steps are stereotyped but the transition speeds between steps are variable. Progenitor-associated transcription factors delay T-lineage differentiation even while Notch-induced transcription factors within the same cells push differentiation forward. Progress depends on regulator cross-repression, on breaching chromatin barriers, and on shifting, competitive collaborations between stage-specific and stably expressed transcription factors, as reviewed here.

T-cell development provides an excellent model system for studying lineage commitment from a multipotent progenitor. The intrathymic development process has been thoroughly studied. The molecular circuitry controlling it has been dissected and the necessary steps like programmed shut off of progenitor genes and T-cell genes upregulation have been revealed. However, the exact timing between decision-making and commitment stage remains unexplored. To this end, we implemented an agent-based multi-scale model to investigate inheritance in early T-cell development. Treating each cell as an agent provides a powerful tool as it tracks each individual cell of a simulated T-cell colony, enabling the construction of lineage trees. Based on the lineage trees, we introduce the concept of the last common ancestors (LCA) of committed cells and analyse their relations, both at single-cell level and population level. In addition to simulating wild-type development, we also conduct knockdown analysis. Our simulations predicted that the commitment is a three-step process that occurs on average over several cell generations once a cell is first prepared by a transcriptional switch. This is followed by the loss of the Bcl11b-opposing function approximately two to three generations later. This is when our LCA analysis indicates that the decision to commit is taken even though in general another one to two generations elapse before the cell actually becomes committed by transitioning to the DN2b state. Our results showed that there is decision inheritance in the commitment mechanism.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (T _regs ) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and T _regs , with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2–induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type–specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type–specific fashion.